ELISA
Introduction Enzyme-linked immunosorbent assay (ELISA) is a method for detecting proteins via their interaction with an antibody. This interaction is usually detected with a colorimetric change in which fluorescence or optical density is the output. Typically performed in a 96-well format, the proteins or antibodies will generally passively bind the polystyrene plate. The antibody used in the assay has a certain specificity for a particular unknown amount of antigen presented on the protein. Once the antigen is immobilized (bound to the plate) detection antibody is added and allowed to bind forming an Ab-Ag complex. This immobilizes the entity and makes the reaction easily readable and detectable. Immobilization also ensures that proper washing away of non-specifically bound materials is performed. The most commonly used enzymes to catalyze the reaction are horseradish peroxidase (HRP) or alkaline phosphotase (AP). Other enzymes are sometimes used although, because of limited production of substrate, HRP and AP are the preferred options among most applications. Limiting factors for choice of substrate in the assay depend on the sensitivity of the reaction and the instrumentation available for signal detection (spectrophotometer, fluorometer or luminometer) (1) (2). What's it used for? ELISA is a powerful analytical technique that is used to detect specific antigens (or antibodies) found on proteins of interest. ELISA has been used in a variety of diagnostic strategies such as: HIV testing (3), West Nile virus , malaria, Chagas disease and many others (2). The utilization of ELISA for diagnostic purposes has gained significant notoriety in recent years. ELISA is also known to be a powerful tool for toxilogical screens of certain classes of drugs. ELISAs are also often used in the food industry for the potential detection of food allergens such as peanuts, milk, walnuts and eggs. Wigand et al, recently reported on a newly developed ELISA for diagnosing systemic lupus erythematosus (SLE), proving its reliability and efficiency (4) . Double stranded DNA (dsDNA) antibodies (anti-dsDNA) are commonly found in patients with SLE, thus detection of these antibodies can be a useful diagnostic tool for clinicians (according to the American College of Rheumatology). Although, often false positive results (patients with rheumatoid arthritis and/or persons who are L-dsDNA-Ab positive) are provided via basic ELISA formats. The newly described ELISA utilizes recombinant dsDNA (h-Rek), which immobilizes human recombinant DNA rather than undefined genomic or rPlasmid DNA and proved to be more accurate and efficient than its predefined predecessor (4). ELISAs are utilized as a diagnostic tool because of its specificity, cost and commercial availability, quick turn around and ease of use. Clinical laboratories will use ELISA as a primary diagnostic tool to screen the blood or serum or even tissue of an individual, and upon a positive result, more testing is then required to validate the findings. Origins and History Prior to the creation of ELISA, radioimmunoassays were the only way of conducting an immune-based reaction assay. Due to the health hazards connected with radioimmunoassays, a safer alternative was sought after. Linking of a substrate to an antibody was an independently developed method by Avrameas and Pierce (5). Fixing (immobilization) of the antibody or antigen to the surface of the container (dish or plate) was also independently developed by Jerker Porath in 1966 (6) . In 1971, two researcher teams, Perlmann and Engvall at Stockholm University in Sweden, and Schuurs and van Weeman in the Netherlands, published papers independently of each other that utlized the aforementioned information which birthed the method EIA/ELISA (7) (8). References #http://www.piercenet.com/method/overview-elisa #http://en.wikipedia.org/wiki/ELISA #http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm #Wigand R, et al. (1997) of dsDNA antibodies in diagnosis of systemic lupus erythematosus--comparative studies of diagnostic effectiveness of 3 ELISA methods with different antigens and a Crithidia luciliae immunofluorescence test. Z Rheumatol 56(2):53-62 #Lequin RM (2005) Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry 51(12):2415-2418 #Wide L, Porath J. Radioimmunoassay of proteins with the use of Sephadex-coupled antibodies. Biochem Biophys Acta 1966; 30:257-260 #Engvall E & Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8(9):871-874 #Van Weemen BK & Schuurs AHWM (1971) Immunoassay using antigen—enzyme conjugates. FEBS Letters 15(3):232-236